Stress-Activated Chaperones: A First Line of Defense

Abstract

Proteins are constantly challenged by environmental stress conditions that threaten their structure and function. Especially problematic are oxidative, acid, and severe heat stress which induce very rapid and widespread protein unfolding and generate conditions that make canonical chaperones and/or transcriptional responses inadequate to protect the proteome. We review here recent advances in identifying and characterizing stress-activated chaperones which are inactive under non-stress conditions but become potent chaperones under specific protein-unfolding stress conditions. We discuss the post-translational mechanisms by which these chaperones sense stress, and consider the role that intrinsic disorder plays in their regulation and function. We examine their physiological roles under both non-stress and stress conditions, their integration into the cellular proteostasis network, and their potential as novel therapeutic targets.

Keywords: molecular chaperone; oxidative stress; protein aggregation; protein unfolding; proteostasis.

Figure 1. Stress-Activated Chaperones Ensure Bacterial Survival

Bacteria are exposed to extremely fast acting stress conditions that have evolved as part of the mammalian host defense. Ingested gram-negative bacteria that colonize the intestine need to survive the highly acidic environment of the stomach. Their porous outer membrane (dashed line) allows the free passage of small molecules such as protons causing the sudden acid-induced unfolding of periplasmic proteins (red shaded area). To deal with these stress conditions, Gram-negative bacteria employ the periplasmic chaperones HdeA and HdeB (grey), which undergo rapid acid-induced unfolding and dissociation, activating their ATP-independent chaperone function. Chaperone-active HdeA/B bind unfolding proteins (red), maintain their solubility and prevent the accumulation of toxic protein aggregates. Upon return to neutral pH (green shaded area), HdeA mediates client protein refolding concomitant with its own refolding. Another mammalian host defense involves the production of high levels of reactive oxygen or chlorine species (ROS/RCS), such as H₂O₂ and HOCl, respectively at gut epithelia and in macrophages. ROS/RCS-mediated side chain modifications cause protein unfolding and aggregation in the cytosol (red shaded area within solid line). In addition, ROS/RCS cause a decline in cellular ATP levels, and directly inhibit canonical ATP-dependent chaperones. In bacteria, ATP is largely rerouted to polyphosphate, which functions as chaperone itself. To cope with this stress, bacteria employ redox-regulated ATP-independent chaperone such as Hsp33 and RidA (blue), which sense ROS/RCS through disulfide bond formation or N-chlorination of amino acid side chains, respectively. Chaperone-active Hsp33/RidA bind unfolding proteins, prevent their aggregation and maintain their solubility. Once reducing, non-stress conditions and ATP levels are restored, canonical chaperones release and refold the unfolded client proteins bound to Hsp33. So far it remains unclear whether RidA can transfer its substrate to canonical chaperones for refolding.

Figure 2. Analogies between pro-and eukaryotic redox-regulated chaperones

Hsp33 in bacteria and the structurally unrelated tail-anchored protein insertion factor Get3 in yeast turn into effective ATP-independent chaperones during oxidative stress conditions. Under reducing non-stress conditions, both proteins are chaperone-inactive. Hsp33, a monomer when reduced, contains four redox sensitive cysteines (indicated with stars), which are located in the C-terminal redox-sensing domain (yellow). The cysteines are arranged in a C-X-C and C-X-Y-C motif and coordinate one zinc ion. Zinc binding confers stability to the C-terminus as well as an adjacent flexible linker region (green), which nestles onto the N-terminal domain of Hsp33 (blue). Get3, a dimer when reduced, also contains four redox sensitive cysteines arranged in a C-X-C and C-X-Y-C motif (stars) and coordinates zinc (grey sphere). In contrast to Hsp33, however, only the C-X-C motifs of the two monomers coordinate zinc, which stabilizes the reduced dimer. The two other cysteines are located in close vicinity to the ATP binding pocket of the ATPase domain (light green) of Get3. Upon exposure to oxidative stress, each protein forms two intramolecular disulfide bonds, connecting the two neighboring cysteines (red star) within the respective motifs. This oxidative disulfide bond formation causes zinc release in both proteins, and massive structural rearrangements. Oxidative activation of Hsp33 involves significant unfolding of the C-terminal redox-sensing domain and the flexible linker region, and is accompanied by dimerization. In Get3, structural rearrangements appear to affect the ATPase domain and alpha-helical subdomain (blue) and leads to the formation of tetramers and higher oligomers with no significant ATPase activity. Both oxidized Hsp33 and Get3 bind tightly to unfolding protein intermediates and prevent their aggregation in an ATP-independent manner. Upon return to non-stress condition, thiols in both chaperones become reduced and the structural rearrangements are reversed. Unfolded substrate proteins bound to Hsp33 are transferred to the DnaK/DnaJ/GrpE system, which promotes refolding to their native structures. The fate of Get3's client proteins remains to be elucidated.

Figure 3. Peroxiredoxins - A Multi-stress Sensing Dual-Function Protein Family

Members of the peroxiredoxin (PRX) family (blue) effectively detoxify peroxide and other reactive oxygen and nitrogen species (green shaded area). As part of the oxidation/reduction process, PRX cycles between reduced decamers and oxidized dimers. Members of the PRX family have been found to switch into potent ATP-independent chaperones once specific protein-unfolding stress conditions are encountered. Leishmania infantum peroxiredoxin mTXNPx, for instance, specifically senses heat stress, whereas Schistosoma mansoni SmPrxI's responds to pH stress and Tsa1 from Saccharomyces cerevisiae is activated by high levels of ROS. The mechanism of stress sensing depends on the respective stress conditions (please see text for details) but the structural changes that the PRX members undergo appear to be similar. They include the formation of chaperone-active decamers and higher oligomeric states, which show increased exposure of hydrophobic surfaces, and decreased peroxiase activity. The chaperone-active peroxiredoxins bind unfolding proteins and hence prevent the formation of cytotoxic protein aggregates. While the binding of unfolding client proteins in the center of the ring-like structure has been shown for mTXNPx, it is unclear where the other PRXs bind their client proteins. mTXNPx interacts with canonical chaperone systems to release the client proteins and support their refolding. Stress-specific peroxiredoxins in parasites appear to be necessary for parasitic organisms to successfully propagate in the mammalian host.